The long-term objectives of this proposal are to understand how the host recognizes and discriminates between microbes that live in distinct cellular compartments, elicits a pathogen-specific immunological response, and generates protective immunity. Listeria monocytogenes is an intracellular bacterium that activates two distinct sets of transcriptional responses in macrophages: a MyD88-dependent response initiated during cell surface and vacuolar recognition and an IRF3-dependent response initiated during cytosolic entry. This bacterium will be used as a model microorganism to determine what role the IRF3- dependent cytosolic response plays during acute infection and long-term immunity. L. monocytogenes mutants that have altered expression of multidrug resistance transporters and consequently induce varying magnitudes of cytosolic gene expression will be exploited to determine the effect of the cytosolic response during infection. The first specific aim will examine the contribution ofthe cytosolic response during acute L. monocytogenes infection in the absence of the TLR-mediated responses. The goal of the second specific aim is to elucidate whether the cytosolic response is involved in dendritic cell maturation and T cell priming. Finally, the third specific aim will address whether the cytosolic response contributes to the generation of T- cell mediated protective immune responses. Uncovering the role of the IRF3-mediated cytosolic response to L. monocytogenes could reveal a new innate immune mechanism important for the generation of protective immunity, knowledge that would be instrumental for vaccine development. The major challenge of vaccine design is how to elicit an immune response that establishes long-term protective immunity against a specific pathogenic microorganism. The research outlined in this proposal will examine how the early inflammatory response to infection with the bacterium Listeria monocytogenes leads to the generation of immunological memory that protects against reinfection. This work will contribute to the understanding of how inflammation generates long-term memory and may reveal new insights on methods to develop safe and effective vaccines.